N-acetylglucosaminyl-lysosomal enzyme-1-phosphotransferase is a membrane bound enzyme that is pivotal in equipping lysosomal enzymes with phosphorylated mannose moieties, residues which target lysosomal enzymes to lysosomes. Deficiency of the enzyme results in the inherited disorders Mucolipidosis I and II. Isolation of the enzyme to obtain sequence data for use in cloning has been based on the selective affinity of the phosphotransferase for a conformationally dependent protein determinant shared by all lysosomal enzymes. 70% of the phosphotransferase activity in Hela cell membranes will bind in solution to a recombinant lysosomal enzyme, beta-hexosaminidase B, and that this complex can be isolated with antibody to beta-hexosaminidase B. Use of (35)S labeled membranes demonstrates that the affinity procedure provides a major purification step and suggests that a 67 kDa protein is a component of the phosphotransferase. The feasibility of using the Vaccinia/T7 transient expression system for cDNA cloning of the phosphotransferase has been examined. N-acetyl-neuraminidase is deficient in the inherited lysosomal storage disorders, sialidosis and galactosialidosis. The former disease is thought to arise from mutations in the neuraminidase structural gene, whereas the latter results from a defect in a glycoprotein (protective protein) which appears essential for maintenance of activity of both neuraminidase and beta-galactosidase. All three proteins copurify and are believed to exist as a functional complex within the lysosome. Two approaches are being utilized to clone the mammalian neuraminidase. (1) The complex has been purified from bovine testicular tissue for use in isolation of neuraminidase to obtain sequence data for cloning. (2) A set of degenerate primers, based on a five amino acid sequence motif found in viral, bacterial and trypanosome neuraminidases, along with mRNA from human fibroblasts has yielded several cDNA fragments following PCR amplification. Sequence analysis of the fragments will demonstrate whether an authentic part of human neuraminidase has been obtained for use in screening a cDNA library.